Forensic Serology PDF

Forensic Serology 6 6.1 INTRODUCTION TO 40 trillion cells with vital nourishment while removing unwanted...

Forensic Serology 6 6.1 INTRODUCTION TO 40 trillion cells with vital nourishment while removing unwanted byproducts (Figure 6.1). It bathes all our tissues in a constant and FORENSICS SCIENCE complex supply of the materials necessary to deliver sustenance, provide protection, and even lend some physical structure to our Forensic Serology, Blood, and bodies. These components must deliver oxygen, recognize, and Immunoassay: The Fluids of Life fight off outside invasion, remove chemical byproducts, trans‑ port cellular materials, and yet remain fluid (Figure 6.2). Blood Yet who would have thought the old man to have had so must squeeze through the tiniest of capillaries, where individual much blood in him? red blood cells must twist and distort to even go through in sin‑ ([Lady Macbeth] William Shakespeare, 1564–1616) gle file, and without requiring so much fluid backpressure as to damage other sensitive tissues and vessels. When blood flow is interrupted for even a very brief time, our cells quickly wither and die. When a breach in our blood delivery system occurs, such as through injury or illness, components within the blood LEARNING GOALS must react very quickly as first responders to try to stop blood AND OBJECTIVES and pressure loss and to effectively repair the breach. In short, blood truly is a miraculous substance, wonderfully suited to the many tasks it is called upon to perform. The study of body fluids, especially blood, both regarding its But blood is also often a very visible part of criminal biochemical composition and its physical fluid properties, can events. A hundred years ago, the legal world was desper‑ yield information of importance to forensic investigations. ately seeking some unique marker that could unambiguously After completing this chapter, you should be able to: tie a particular individual to a biological sample collected at a crime scene. The best technique at the time for doing this, Understand what is meant by the term serology. Describe how blood functions in our bodies and rec‑ ognize its various components. Explain what is meant by presumptive and confir‑ matory tests. Summarize how blood can be detected and identi‑ fied as human blood. Discuss how different immunoassays work. Identify the various blood types and illustrate what this means biochemically. Explain what is meant by hereditary patterns of blood types. AU: As per style, Discuss what is meant by blood pattern analysis. please Illustrate how events can be understood through provide complete blood pattern analysis. refer- Identify how other body fluids can be used in foren‑ ence details sic science. in list for the sources FIGURE 6.1 About 60,000 miles of blood vessels, including present 6.1.1 Introduction arteries, veins, and capillaries, carry blood throughout the body in all fig- ures and to infiltrate our tissues, thereby supplying them with neces- update Blood is our most precious life‑giving fluid. It flows continuously sary nutrients, exchanging gases and performing a host of vital name functions. and year in each person through a closed‑loop of nearly 60,000 miles of format in arteries, veins, and capillaries, efficiently providing our nearly sources. Source: Shutterstock.com. 154 DOI: 10.4324/9781003183709-8  6 Forensic Serology 155 the laboratory can further provide a dazzling amount of infor‑ mation from these easily collected specimens. Blood analysis remains one of the most powerful tools for understanding samples of forensic interest. Blood assays can often be performed in a matter of minutes using relatively inexpensive instruments and techniques. These analyses may serve as a first screen for whether or not to employ the more instrument‑intensive DNA typing or, in some cases, to pro‑ vide independent laboratory corroboration for the conclusions derived from the DNA data. Equally important, however, is the biochemical analysis of blood and the things that it carries, since this may provide information for forensic medical and toxicological investiga‑ tions not obtainable in any other fashion. Understanding some key features about blood chemistry is, therefore, important FIGURE 6.2 Red blood cells, called erythrocytes, are the disc‑­ shaped cells that primarily transport gases throughout the body. not just for personal identification and crime scene analysis but also in determinations such as cause of death, post‑mor‑ Source: Shutterstock.com. tem interval, the presence of drugs and alcohol, and many others. The concepts of blood chemistry discussed in this chapter will, therefore, be fundamental in considering sev‑ fingerprinting, had both its successes and serious limitations eral other chapters including forensic medicine (Chapter 8), (Chapter 7). Many times, interpretable fingerprints were not forensic toxicology (Chapter 13), and forensic psychology found at the crime scene, while biochemical tissues and fluids (Chapter 19). were abundantly available. After a landmark discovery by Karl Blood and other biological fluids are frequently found at Landsteiner in 1901, however, attention turned to the analysis crime scenes in relatively large amounts, especially in crimes of blood to hopefully provide such vital connections between of violence. Because of their seriousness, these are also usually evidence and suspects. Through careful scientific investi‑ the crimes for which we must have the best and most detailed gation, blood analysis quickly reached an impressive level information. of detail that was able to provide the needed links between Besides information about the chemical composition of a forensic samples, often with millions‑to‑one odds of random blood sample, the patterns and locations of fluid samples can matches. This analysis remained the gold standard for forensic provide investigators with a great deal of information about identification until DNA typing became the method of choice how a crime might have been committed and the sequence of in the late 1980s. Today, blood analysis still provides an enor‑ events that occurred. mous amount of unique information to a forensic investigation, Serology is broadly defined as the study of serums, or body shedding light on both questions of “who” and “how.” fluids and liquids (the plural for serum can be either serums or Since Landsteiner’s discoveries, advances in understand‑ sera). While there are many different types of serums in the ing biological fluids have coupled with the development of human body that we will touch upon in this chapter, we will new biochemical techniques to produce an arsenal of compel‑ focus most of our attention here on the forensic application of ling forensic probes. Since blood is such an integral part of blood analysis: both biochemical and physical. our immune system, immunological methods in blood analy‑ In this chapter, we will begin by examining the basic fea‑ sis have become centrally important. Simple but vital ques‑ tures of blood and body serums and how such detailed infor‑ tions such as “is this blood,” “is this blood human,” and “what mation can be used in forensic investigations. We will also biochemical markers may be found in the blood” can now be explore how the physical patterns of blood and other biologi‑ very quickly answered, even at the crime scene itself. More cal fluids at a crime scene can provide information about the sophisticated immunological studies of a forensic sample in sequence of events leading to the observed crime scene. BOX 6.1 CASE HISTORY: LUDWIG TESSNOW In early July of 1901, two young brothers, aged 6 and 8, did not return home after a day of playing together on Rugen Island in northern Germany. The next day, a full‑scale search was mounted when their dismembered bodies were found spread over a wide area of the island. A local carpenter remembered seeing the boys talking with another local resident, Ludwig Tessnow, early on the day that they disappeared. Investigators went to Tessnow’s home where they found his freshly laun‑ dered work clothes with unusual stains that could have been bloodstains. Tessnow, however, quickly explained that these were wood dye stains, not unusual for a carpenter of the time. In addition, a local farmer identified Tessnow from a line‑up as the man who was seen dismembering seven of his sheep and tossing their legs around a field. When considering the case, an alert local magistrate remembered that, in another case, three years earlier and 300 miles to the east, Tessnow had 156 Introduction to Forensic Science also been a prime suspect in a dismembering and had used the same reasons to explain similar stains found on his cloth‑ ing then. In that earlier case, two young girls of Osnabruck, Germany had been found dismembered in a similar fashion to the two Rugen boys. The problem, however, was that there was no other evidence connecting Tessnow with any of the crimes, and it was impossible at the time to determine if the stains on his clothing came from wood dye, sheep’s blood, or human blood. The answer to this dilemma came from Prof. Paul Uhlenhuth at the nearby University of Greifswald. Dr. Uhlenhuth had just developed a new test that could be used to differentiate human blood from other stains, even from other types of animal blood. In his work, he had injected hen’s blood into rabbits and then isolated the serum (liquid portion) from the rabbit’s blood. When this same rabbit serum later came in contact with hen’s blood, a reaction occurred, causing a solid precipitate to immediately form. When the blood from other animals was placed in the rabbit serum, however, no reaction was found. He further demonstrated that he could use similarly prepared animal serums to distinguish uniquely the blood of one animal from all other species of animals, including humans. Thus, when human blood was injected into another animal and the serum from that animal was isolated, now called human anti‑serum, it reacted only with human blood and none other to form a solid. In the Tessnow case, Prof. Uhlenhuth examined the overalls taken from Tessnow and determined, using his human anti‑serum, that besides the wood dye, the clothing conclusively contained many traces of both human and sheep’s blood. Tessnow’s explanation immediately fell apart, and he was quickly convicted at trial and executed in 1904 for his crimes. This case demonstrated the first use of an immunological test to determine the presence of human blood in a forensic sample. However, the significance of this case goes well beyond the Tessnow case: it served to strengthen the importance of forensic science in the courtroom and to place science as a powerful ally in crime detection and legal prosecution. A modification of this test is still employed diagnostically today 6.2 BLOOD AND IMMUNOASSAY as a colon cancer screen to determine if invisible, minute quanti‑ ties of blood (called “occult blood”) are present in fecal materi‑ als. The forensic problem with this test is that it gives many false 6.2.1 Background and History positives and cannot distinguish human from animal blood. Another test, also still in use, is called the Kastle‑Meyer test. of Blood Analysis in This method uses a chemical called phenolphthalein that, when Crime Detection mixed with hydrogen peroxide and blood, changes in color from colorless to pink. Like the Guaiacum test, the Kastle‑Meyer test Blood consists of an amazing collection of cellular, biochemi‑ cannot discriminate between human blood and animal blood cal, inorganic, and liquid components that perform an enor‑ and gives false positives from potatoes, horseradishes, and a mous array of life‑sustaining functions. It is the fragile link number of other natural materials. Other color‑change tests have that nourishes, supports, and defends our cells. It is also all too been developed, but all suffer from similar problems. Criminals often shed in the commission of crimes and, therefore, leaves could simply claim that the positive test arose from the fact that a trail of valuable evidence to be considered. they had come into contact with animal blood, raw meat, or even For centuries, investigators have longed for the ability to that they had eaten horseradish. The problem was that these learn the secrets hidden in the blood found at crime scenes. claims could not be easily disproven. Even simple questions of whether or not a stain was really Around the turn of the 19th century, a series of discover‑ blood remained elusive until the 20th century. Simple observa‑ ies began to change the face of blood analysis. In 1879, Louis tions such as the color, smell, and texture of a suspected blood‑ Pasteur was working on understanding cholera in chickens. stain are unreliable indicators. Dried blood, for example, may He noticed that when he accidentally injected a deteriorated appear as red, brown, or even greenish‑yellow depending on cholera sample into some chickens, they became sick but ulti‑ the sample’s age and history. The difficult problems of blood mately recovered fully from this usually fatal disease. When analysis even surfaced in Arthur Conan Doyle’s 1887 book “A he later tried to reuse these recovered chickens in his work, Study in Scarlet” when Sherlock Holmes, the quintessential he found that he could not infect them with even the strongest detective, complains about the inadequacies of then‑existing strains of cholera. These chickens had developed an immunity blood tests and demonstrates his own “new” procedure. to cholera. He then extended his work in a similar fashion to By the late 1800s, a series of chemical tests had been devel‑ include animal immunizations for anthrax and rabies. Pasteur’s oped that could provide an indication of whether blood was work, in which a weakened or “dead” strain of a disease is first present. Most relied upon an observable color change when a injected to cause immunity to the full‑strength contagion, actu‑ standard reagent came into contact with a suspected blood sam‑ ally built upon the much earlier work of Edward Jenner who, ple. One of the first of these was the Guaiacum test, in which a in 1796, used cowpox injections in people to give them immu‑ plant extract turned blue when brought into contact with blood. nity from the terrible smallpox disease (Figure 6.3). Jenner  6 Forensic Serology 157 FIGURE 6.3 Edward Jenner (1749–1823), a British physician, is shown in the painting inoculating a child. Jenner first developed a vaccine for the often‑fatal smallpox infection. He investigated common folk stories about how people who had survived the milder cowpox infection became immune to smallpox. In 1796, he inoculated a healthy child with pus from a cowpox sore on a dairymaid’s finger. Weeks later, he exposed the child to the smallpox contagion (definitely not something practiced today in medical research), but the child did not develop the disease. This immunization process was named vaccination after the name of the cowpox virus (variola vaccinia). Smallpox was declared extinct outside of laboratories in 1980. coined the word vaccination for the process from the Latin name for the cowpox virus (variola vaccinia). Later, in 1892, Emil von Behring found that animals exposed to a toxin of diphtheria developed a resistance to the disease in the blood. A huge advance for blood forensic analysis finally came in 1900 when the Uhlenhuth serum‑based test, first demonstrated in the Tessnow case (see insert box), was found to be specific for a particular type of blood – human blood. This new “precipitin test” could readily distinguish human blood from all others. In fact, specific tests were later developed to distinguish between blood samples for many different species of animals based on the precipitin test. But now that it was possible, using the precipitin test, to establish that a sample truly contained human blood, the next Clysmatica nova 1667 Attempted early blood transfusion important question became “whose blood?” Was it even pos‑ from lamb to man sible to say with any certainty that a particular blood sample originated from a given person? FIGURE 6.4 Historical Blood Transfusions. (Left) 17th‑century The roots to answering this question lie in medieval times. artwork of a patient during a blood transfusion, taken from the For centuries, people had sought for ways to quickly replenish 1667 book by Johann S. Elsholtz (Clysmatica nova), and (Right) the blood supply of people who had lost significant quantities 17th‑century artwork showing Richard Lower (1631–1691) trans- of blood through accidents, injuries, or diseases. The main idea fusing blood into a man’s arm from a lamb. The tubes used to transfer the blood are shown at the top left. These transfusions was to take blood from a healthy person or animal and give it were often fatal for the patients due to blood incompatibilities, to the injured person to restore some of the lost blood until leading to coagulation and cell rupture. In 1901, Karl Landsteiner their body could replenish the supply itself (Figure 6.4). While discovered the ABO blood groups. After the discovery of other this was occasionally successful, it more often resulted in the blood groupings (e.g., M, N, and P groups) in the 1920s, and the death of the recipient. Sparked by these failures, human blood Rhesus factor in the 1930s, transfusions became generally safe transfusions were banned by the early 1700s across much of to practice. 158 Introduction to Forensic Science Europe. Experiments with animals, however, showed that it Serology is the area of science that deals specifically with was sometimes possible to transfer blood from one animal to the study of serums (body fluids), such as blood, saliva, urine, another without significant problems. The important question semen, blisters, sweat, and others. However, it often focuses on then became why this process was not similarly successful in blood serum since that is the most abundant serum in the body. human patients? Serology also deals with antibody‑antigen reactions within the The first answers to why fatalities happened in humans blood, and clinically it may involve the diagnostic identifica‑ came in 1875 from the work of Leonard Landois when he tion of antibodies found in body fluids. observed that when blood from two different humans was Immunology is the broad branch of science that deals mixed, the cells often clumped together and sometimes were with aspects of the immune system. This huge field is broken even ripped into pieces. This “clumping” process resulted in the into several subfields including clinical immunology, diagnos‑ formation of very many blood clots throughout the body, often tic immunology, immunotherapy, evolutionary immunology, leading to kidney damage, shock, cardiac arrest, and, ultimately, and others. In diagnostic immunology, the unique selectivity to death. In 1901, Karl Landsteiner provided the first landmark of the interactions between antibodies and antigens has led to understanding of why this happens and, most importantly, how the development of an array of detection methods for a variety to avoid it from happening during transfusions. He discovered of substances that are of interest to forensic investigators. For what we know today as the ABO system of blood typing. He example, immunoassay tests for blood types, microbes, drugs, found that people had a variety of possible proteins associated toxins, and diseases have revolutionized the detail of forensic with their red blood cells. His work allowed the determina‑ information available. These tests are often fast, inexpensive, tion of the incompatibility of mixtures of human blood before highly selective (with few false positives and negatives), and a transfusion took place, saving countless lives and setting the extremely sensitive for the target component. stage for the forensic individualization of blood samples. Fluid dynamics, located at the intersection of physics and In the 1920s, researchers found that blood type can often engineering, deals primarily with the behavior of liquids and be determined from other body fluids besides blood, mean‑ gases in motion. In blood analysis, it focuses on how blood ing that an actual blood sample was not required to determine flows and may involve calculations of density, velocity, surface blood type. In 1949, it was learned that the gender of an indi‑ tension and viscosity. The field may also consider questions vidual could also often be determined from the presence of a such as how blood flows within the body and the mechanisms characteristic structure (Barr body) within white blood cells. of how it can be spread around a crime scene. Since then, we have learned how to glean increasingly individ‑ Blood pattern analysis considers bloodstains found at a ualized information from blood, rather than just class evidence. scene and attempts to provide an understanding of how they But can additional, forensically valuable, information be were formed. It brings together aspects of chemistry, physics, determined from blood – such as a physical record of how a fluid dynamics, and other disciplines to help provide this infor‑ violent crime took place? References as far back as the Bible mation. Questions such as the speed of the weapon causing the and even before describe the importance of blood in under‑ injury, the relative location of the victim and assailant during standing unobserved but violent events (Gen. 4:10: “Your the attack, the order of attacks, and the physical characteristics brother’s blood cries out to me from the ground”). In recent of the weapon used in the attack are among the answers sought years, the science of blood dynamics and blood pattern analy‑ from this type of analysis. sis has developed into an integral part of investigations involv‑ In order to understand more fully how blood analysis can ing blood evidence. provide such information to investigators, a deeper understand‑ Thus, blood can provide us with a wealth of forensic ing of the nature and properties of blood itself is first required. insight – victims and their assailants can speak clearly to investigators through the blood record. Blood evidence can tell us if the sample is human or animal, it can lead us to the 6.2.3 Blood Chemistry individual whose blood it is, it can tell us about diseases in the blood donor, it can provide a trace of lineages, and it can even Blood is composed of many different components, each with a tell us about the events involved in the distribution of blood function to perform. In this section, we will describe the most around a crime scene. important features of these components and discuss how they function in living systems. For forensic purposes, red blood cells and blood plasma, described below, are probably the most 6.2.2 General Definitions important types of evidence. From these two components, a complete blood typing profile can be made. The field of blood and bloodstain pattern analysis encompasses several well‑established areas including serology, immunol‑ 6.2.3.1 Liquid Components of Blood ogy, pathology, and fluid dynamics. Each has information to contribute that completes a picture of the people and events Blood is medically classified as a circulating tissue in the body that led to the observed scene. that comprises about 7% of our total weight, or about 4–6 L  6 Forensic Serology 159 FIGURE 6.5 Three different blood products: the unit on the left shows whole blood containing red blood cells, white blood cells, and platelets suspended in a liquid plasma, the unit in the center contains a concentrated solution of platelets, used to aid in blood clotting, and the unit on the right contains blood plasma. Source: Images courtesy Shutterstock.com. (four to six quarts). Maintaining a normal blood volume is criti‑ blood, the greater the internal friction. The result of this is that cal for maintaining life functions. The loss of only about 10% blood is more resistant to flow than water but is still a relatively of our blood volume causes an increase in blood pressure and free‑flowing liquid (e.g., olive oil has about 20 times the vis‑ other symptoms. Loss of 25–30% of the effective blood volume, cosity of blood). The forensic implications of this will become however, leads to a state of shock with a dangerous drop in blood apparent when we consider bloodstain pattern analysis. pressure and irregular heartbeats. If prolonged or followed by Significant problems arise when the blood supply is inter‑ further blood volume loss, coma and death usually occurs. rupted to our tissues, even for a very brief time. This rapidly Blood consists of a complex mixture of cellular, biochem‑ results in a shortage of the oxygen necessary for respiration ical, and inorganic components, all in a water‑based solution. (cellular energy production) and a buildup of toxic waste prod‑ The liquid portion of blood comprises about 55% of its vol‑ ucts (mainly carbonic acid, CO2, and lactic acid). You might ume and is known as plasma. If the blood is first allowed to experience rapid lactic acid buildup in a limited way as a burn‑ clot, however, followed by removal of the solids by centrifu‑ ing sensation when vigorously exercising – at these times our gation (rapid spinning of the sample), a slightly yellow solu‑ bodies cannot deliver enough oxygen to its tissues for sufficient tion known as blood serum is obtained. The main difference respiration to occur so it uses another mechanism to produce between plasma and serum is that the serum contains none energy that ultimately generates lactic acid. When the blood of the clotting factors found in plasma (principally fibrinogen supply is cut off to a tissue or organ, it is medically referred and platelets). These factors are effectively removed when the to as ischemia and, unless rapidly corrected, usually results in blood clots and the liquid portion is separated from the clotted tissue damage and cellular death. solids. The three types of blood solutions often used medically, including plasma, are shown in Figure 6.5. 6.2.3.2 Cellular Components of Blood The blood plasma rapidly carries nutrients, such as glu‑ cose, amino acids (protein building blocks), lipids, salts, and There are three primary types of cells found in whole blood: fatty acids to all the cells in the body at up to ~12 ft/s (~3.6 m/s). red blood cells (erythrocytes), white blood cells (leucocytes of It consists of about 92% water, 8% blood proteins, and very various types), and platelets (thrombocytes). Drawings of these small amounts of other compounds. Also contained within three cell types are shown in Figure 6.6. the plasma are important “messenger” compounds, such as Red blood cells (RBCs) account for about 96% of the cel‑ hormones, that provide regulatory and signaling information lular portion of blood and appear as flexible, disc‑shaped cells for the body. The blood also serves to very effectively regulate that are concave on both sides. Their primary function is to body temperature, removing excess heat from overheated areas carry oxygen from the lungs to the cells and return waste carbon and providing more warmth to cold regions. For example, after dioxide from the cells back to the lungs. The red color comes you’ve been outside on a cold day, your body sends an increased from the main chemical component of RBCs, the oxygen‑car‑ blood supply to the exposed tissues, seen as a blush, to help rying protein‑iron complex called hemoglobin, which accounts restore proper temperature. Our blood is also slightly basic for about 90% of the dry weight of a mature RBC. The unusual (alkaline), which helps our bodies maintain a proper acid‑base concave disc shape of RBCs arises because this is the most effi‑ (pH) balance. Blood carries immunoglobulins (antibodies) and cient shape for gas exchange between the cell’s interior, where blood‑clotting factors (fibrinogen) to sites where they’re needed the hemoglobin is located, and its environment. When fully to ward off infection and repair damaged tissues. oxygenated, blood is bright red (absorbs mostly blue light), but Whole blood has about the same density as water (1.06 g/ when deoxygenated and loaded with carbon dioxide, it appears mL) but about three times the viscosity (resistance to flow), as a darker red color. A common misconception is that deoxy‑ meaning that the internal friction of blood is significantly genated (venous) blood is actually blue. This is, however, an higher than water. Generally, as more cells are found in the “illusion” based on the fact that when white light, containing all 160 Introduction to Forensic Science FIGURE 6.6 Human blood cells: (Top) a drawing illustrating the different types of blood cells, and (Bottom) a drawing showing the various types of arterial blood cells in plasma. Red blood cells are disc‑shaped and contain hemoglobin, a chemical that combines reversibly with oxygen, allowing them to transport oxygen from the lungs to the tissues. Platelets are produced in the bone marrow and are involved in the process of blood clotting. White blood cells are involved in the human immune system response. Source: Image Sources courtesy Shutterstock.com. FIGURE 6.7 Scanning electron micrograph (SEM) showing fetal blood stem cells. Stem cells are pluripotent, meaning that they are able to differentiate into any type of blood cell through a process called hemopoiesis. Source: Shutterstock.com. visible wavelengths, shines on our skin, the longer wavelength a healthy adult male and between 35% and 45% for a female. red light can easily travel through the skin to be absorbed by the Typically, there are about 200–400 million RBCs in every hemoglobin, while the shorter wavelength blue light is mostly drop of our blood. reflected to our eyes by the skin. RBCs are formed, as are most of the cellular components The proportion of blood made up of RBCs is called the of blood, in the bone marrow from stem cells at a rate of about hematocrit, which usually ranges between 40% and 50% in 2 million new cells per second (Figure 6.7). A unique feature  6 Forensic Serology 161 of RBCs is that, as they mature, they expel their nuclei and RBCs pick up the waste carbon dioxide molecules and travel all their organelles, including their mitochondria. This ren‑ via our veins back to the oxygen‑rich lungs where they release ders RBCs useless for forensic DNA profiling. As RBCs have the carbon dioxide before beginning the process all over again. no nucleus or organelles, they cannot repair themselves and, The capacity of RBCs to transport O2/CO2 can be dramati‑ therefore, typically last for only about 120 days on average cally affected by disease or the presence of other chemicals in before they die and are removed from the system by the spleen our bodies. In an example all too common in forensic investiga‑ or liver. Other waste products in the blood are also removed tions, a common gas in automotive exhaust, carbon monoxide primarily by the liver and kidneys. (CO), also binds very easily to the heme groups of the RBCs, RBCs function to transport oxygen and carbon dioxide in in fact, to the exclusion of oxygen. Carbon monoxide readily the body primarily by the red protein, hemoglobin (Figure 6.8). displaces oxygen from hemoglobin and forms carboxyhemo‑ Each blood cell contains about 270 million hemoglobin mol‑ globin, a molecule that is 140 times more stable than oxyhe‑ ecules, each molecule equipped with four iron‑based heme moglobin, causing it to bind very strongly and to be released groups – the center of oxygen and carbon dioxide transport. only very slowly. As a result, breathing only 0.1% CO in air for When the blood is in the high‑oxygen environment of the lungs, four hours converts 60% of our hemoglobin into carboxyhemo‑ the heme groups in the RBCs reversibly pick up oxygen mol‑ globin! This means that 60% of our hemoglobin is essentially ecules to become oxyhemoglobin. The oxygen‑rich RBCs then unavailable for oxygen transport, potentially leading to asphyx‑ travel throughout the body via the arteries, releasing oxygen to iation. Luckily, CO binding is reversible, although slowly, and if our cells in the oxygen‑poor environment of our tissues. The the patient can be removed from the CO source in time into an release of oxygen is actually aided by the presence of carbon oxygen‑rich atmosphere, they often can recover fully. dioxide in the cells (a bi‑product of respiration) through a com‑ White blood cells, or leukocytes (shown in Figure 6.9), are plex series of chemical reactions. In return, the oxygen‑poor actually a large and diverse group of cell types that differ from RBCs primarily in that they have nuclei and organelles and perform different functions in our bodies. They are typically round to irregularly shaped cells, accounting for about 3% of the total number of blood cells. Their primary responsibility is to fight off disease and infection and to effect cellular repair. A single drop of whole blood typically contains between 10,000 and 25,000 white blood cells. The shape of these cells can rad‑ ically change, however, in response to activation by chemicals or invaders in the blood. These cells are found in a number of places besides in the blood, including in the lymph system, spleen, and liver. White blood cells are formed in the bone marrow at the rate of about 100,000 new cells per second. Recent studies have suggested that the skin may also play an important role FIGURE 6.8 Hemoglobin: (left) a model of the hemoglobin molecule that transports oxygen around the body within red blood cells, and (right) the heme subgroup in hemoglobin. The hemoglobin molecule has four heme subgroups surrounded by four globular protein chains and coils (each protein subunit, containing one heme unit, is shown in a different color in the picture). Each protein subunit is wrapped around a heme group, FIGURE 6.9 Composite micrograph of T‑cells attacking a can- protecting it from being destroyed by the oxygen it is intended to cer cell. T‑cells are a type of white blood cell (lymphocyte) that transport and allowing it to function properly. The iron atom of develop from stem cells and form a key part of the immune sys- each heme group reversibly binds to oxygen and carbon dioxide tem. Lymphocytes, mainly T‑cells and B‑cells, recognize foreign in the blood to transport these gases throughout the body. antigens and destroy attacking viruses and bacteria. Source: Image on left, Shutterstock.com. Source: Shutterstock.com. 162 Introduction to Forensic Science in white blood cell formation. Occasionally, it may be possible to determine how soon after an injury an organism died by BOX 6.2 BRIEF ON BLOOD CHEMISTRY determining the extent of leukocyte attack on infection (see 1. Blood consists of cellular (e.g., RBCs, white Chapters 8 and 9 on forensic medicine and anthropology, blood cells, and platelets), biochemical (e.g., respectively). proteins, carbohydrates, enzymes, etc.), and Among the many types of white blood cells are neutro‑ inorganic (e.g., salts, vitamins, etc.) components. phils, B‑cells, T‑cells, monocytes, and natural killer (NK) cells. 2. The liquid portion of whole blood is plasma, Neutrophils represent our primary defense mechanism against which, after clotting and removal of particu‑ bacterial infections. Pus, the whitish‑yellow substance formed lates, is called serum. at infection sites, is made up of both living and dying neutro‑ 3. RBCs, comprising 96% of cellular matter in phil cells involved in the fight. These neutrophils break down blood, are responsible for the transport of O2 and release chemicals that kill invading bacteria and also signal and CO2 to and from cells. other white blood cells to join the fight. B‑cells and T‑cells are 4. White blood cells (~3% of cells) are involved responsible for making antibodies and coordinating immune in immune responses. responses, respectively. The natural killer cells roam the body 5. Platelets (~1% of cells) assist in repair and kill cells that do not display the correct “do not kill” signal, mechanisms. often infected or cancerous cells. Finally, monocytes are our cellular “vacuum‑cleaners,” eliminating infected cells. Blood platelets, or thrombocytes, account for about 1% of blood cells and play a particularly important role in blood 6.2.3.3 Other Blood Components clotting and self‑repair processes of blood vessels. Like RBCs, “resting” platelets have no nucleus and are smooth, Blood contains hundreds of other chemical components beyond disc‑shaped cells, although much smaller in size than the those described thus far, including fibrinogen, salts, proteins, RBCs (Figure 6.10). Unlike RBCs, They do contain some glycoproteins, carbohydrates, antibodies, hormones (e.g., insu‑ RNA and other cellular structures. lin, testosterone, estrogen, adrenaline, or epinephrine, etc.), Platelets originate in bone marrow and are very short‑lived, albumin, and dissolved gases. The most common protein in typically lasting only about eight to ten days before they are our plasma is albumin, which is responsible for maintaining removed from circulation by the spleen. Platelets come into a proper fluid balance between our tissues and the rest of our action when they detect a breach in a blood vessel from injury bodies. Blood also contains waste products from cellular reac‑ or disease. They quickly transform in shape and initiate a com‑ tions, viral impurities, cell fragments from immune battles, plex chain of biochemical reactions resulting in the release of invading parasites and microbes, and substances that we add sticky fibrin strands and a clumping together of the platelets to our systems (e.g., drugs, alcohol, poisons, etc.). with other blood cells (Figure 6.10). This action forms a blood vessel “plug” that then contracts to further tighten the seal. 6.2.3.4 Blood‑Based Diseases Aspirin and a variety of other compounds interrupt plate‑ let function, which might not return until new platelets are Diseases found in the blood can also provide useful infor‑ generated. mation in forensic analysis. These conditions can arise FIGURE 6.10 (Left) Image of non‑activated blood platelets (thrombocytes). These platelets are formed from bone marrow cells (megakaryocytes), circulate within the blood, and are responsible for blood clotting. (Center) Image of activated platelets. Activated platelets have long dendritic “fingers” that allow them to clump together if they detect a break in a blood vessel. (Right) A thrombus in the bloodstream, a blood clot formed from activated platelets and fibrin. Source: Images courtesy Shutterstock.com.  6 Forensic Serology 163 FIGURE 6.11 Common diseases found in blood cells: (a) malarial parasites, Plasmodium, form inside red blood cells (Used per Creative Commons Share‑Alike 4.0, International, User: Dr. Graham Beards), (b) medically accurate illustration of sickle cells (Source: Shutterstock.com), (c) Trypanosoma cruzi parasite illustration, a protozoan that causes Chagas’ disease transmitted to humans by the bite of triatomine bug (Source: Shutterstock.com), and (d) HIV vs Normal RBC (shown is a red blood cell diagnostic test for an HIV infection; ‘clumping’ indicates a positive result). Source: Used per Creative Commons Attribution 3.0, Unported, User: CSIRO. from genetic disorders (e.g., sickle cell anemia and hemo‑ 6.2.4 Blood Testing philia), diseases (e.g., leukemia and cancer), viral and bac‑ terial infections (e.g., HIV, SARS‑CoV‑2, and Lyme), and 6.2.4.1 Three Questions of Blood parasitic invasions (e.g., malaria, sleeping sickness, etc.) (Figure 6.11). For example, sickle cell anemia results when So, now that we know more about the basic chemistry of blood the hemoglobin protein in the RBCs has one amino acid and how it functions in our bodies, we can begin to answer changed from the normal variant. People with this disease more fully the questions posed earlier such as “is it blood?”; “is make hemoglobin S instead of the normal hemoglobin A it human blood?”; and ultimately “whose blood is it?” In this molecules. Hemoglobin S carries a smaller negative charge section, we will take these questions one at a time, beginning than hemoglobin A, causing it to clump together more read‑ with “presumptive” blood tests. ily than hemoglobin A when it loses oxygen. This results in the observed sickle‑shaped RBCs (Figure 6.11) that are 6.2.4.2 Question One: Is It Blood? fragile, short‑lived, and inefficient at oxygen transport under lowered oxygen levels. The sickle cells are rigid and sticky, In the late 1880s, investigators were not able to determine if a leading to clogged arteries and dehydration. Carriers of the suspicious stain was blood or something else, especially since sickle cell allele are, however, more resistant to malaria bloodstains can appear as anything from red to green. Fruit because the malarial parasites are killed inside sickle‑shaped juices, dyes, and other compounds can give stains that look blood cells. remarkably similar to bloodstains. To help ascertain whether a Often, traces of chemicals added to a person’s body for stain was indeed a bloodstain, a series of tests were developed medical treatment or as drugs of abuse can be detected in their to provide a quick visual indication of the presence of blood. blood. Identifying these compounds, along with their concen‑ The advantage of these presumptive tests is that they are fast trations and likely sources, can be an enormous aid to an inves‑ and relatively sensitive. The great disadvantage is that they can tigation. This information is often gained through a chemical give false positive readings – a positive reading when blood toxicological study of a person’s blood and will be discussed is not actually present in the sample, but the test says it is. In in Chapter 13. presumptive tests, if a test is negative, the blood is believed 164 Introduction to Forensic Science most likely to be absent, but if the test gives a positive result, colored material by an oxidant, such as hydrogen peroxide then blood is probably, although not definitely, present. Thus, (Figure 6.12). If you’ve ever put hydrogen peroxide on a cut to a presumptive test is an analysis that suggests that blood could disinfect it and noticed that it bubbled profusely, you’ve prob‑ be present in a sample. Today, these tests are used as effective ably observed this same reaction. The bubbling comes from screens to determine whether or not additional, more conclu‑ the release of oxygen gas (O2) due to the decomposition of sive (confirmatory) testing is warranted. A confirmatory test hydrogen peroxide (H2O2) to water and oxygen caused by the is an experiment that can indicate the presence of a particular hemoglobin. The Kastle‑Meyer test, like all other presumptive chemical in the sample, in this case blood, with a very high tests, however, cannot distinguish human blood from animal degree of certainty. blood and can give false positive results from non‑blood com‑ One of the first presumptive tests developed was the ponents also in the sample, such as horseradish and potatoes Guaiacum test, in which a solution turns blue in the presence (or anything that behaves as a biochemical peroxidase, an of a blood sample. Due to significant problems associated with enzyme that catalyzes the oxidation of another compound by the accuracy and ease of the Guaiacum test, a variety of other the decomposition of a peroxide). In fact, the presence of any presumptive color‑change tests were developed, including the naturally occurring peroxidase compound will give a positive benzidine (Adler test), leucomalachite green, and ortho‑tol‑ test but, to an observant analyst, the color change seen from idine tests. While these procedures still retain some limited these “false positive” compounds is often noticeably slower to use, they have fallen out of general use because they involve develop than for a blood sample. These tests are often suc‑ either toxic, cancer‑causing, or difficult‑to‑handle chemicals. cessful on dried and old samples, including a recent report of These tests have now mostly been replaced by the tetrameth‑ a successful blood test from the uniform of a wounded officer ylbenzidine (TMB) and Kastle‑Meyer tests (Figure 6.12). In from the War of 1812. the Kastle‑Meyer procedure, the most commonly employed At crime scenes, it may be important to find the locations presumptive field blood test, a suspected sample is collected of suspected blood residues that cannot be seen by the naked on a swab with alcohol, and a few drops of phenolphthalein eye. For this purpose, two reagents, called luminol and fluo‑ are added followed by a few drops of hydrogen peroxide rescein, are usually employed. While their modes of action are (H2O2). If blood is present, the swab immediately turns pink quite different, both reagents provide a visible glow where very (Figure 6.13). One important advantage of this test is that it is minute residues of blood are found. The use of these reagents typically non‑destructive, allowing further tests to be run on can reveal patterns, such as shoeprints and drag stains, that the sample if needed, including DNA analysis. would otherwise be undetectable to the unaided eye. These presumptive color‑change tests rely on the cata‑ In the luminol procedure, a weak solution of the chemical lytic behavior of the heme group in blood to catalyze the oxi‑ luminol (with an activator) is sprayed where the blood is sus‑ dation of a colorless material, such as phenolphthalein, to a pected. The iron from trace amounts of hemoglobin catalyzes FIGURE 6.12 Kastle‑Meyer test involving the catalytic oxidation of the colorless phenolphthalein indicator to pink by the heme groups in hemoglobin.  6 Forensic Serology 165 when illuminated by UV light. Fluorescence is quite different from the chemiluminescence process of luminol and occurs when a molecule absorbs light at one wavelength and then emits light at a different wavelength (Chapter 12). With fluo‑ rescein, the oxidized molecule absorbs light in the ultraviolet region that our eyes cannot see and then emits light in the vis‑ ible region, producing a blue glow. Sometimes, it is also desirable to enhance weakly visible bloodstains, for example, to make bloody footprints, finger‑ prints, and other patterns more visible. Luminol and fluores‑ cein can be used for this, but they tend to blur the pattern’s detail. Instead, a solution of a dye, such as Leucocrystal violet, can be used very effectively. It is an extremely simple proce‑ dure; simply spraying the reagent on the stain immediately produces a permanent dark purple color. Similar results can also be obtained using a variety of other dyes (e.g., Hungarian red, amido black, Crowle’s Stain, Coomassie Blue, etc.). All the tests described so far are presumptive tests, sug‑ gesting whether or not blood is present in the sample, helping to decide whether to move to more detailed studies (confirma‑ tory tests). A variety of confirmatory tests have been developed over the past century to determine conclusively if a stain con‑ tains blood. One group of these tests is called “crystal tests” because, when treated with a specific chemical, blood uniquely forms identifiable crystals under the microscope. For example, in the Takayama test, a solution containing pyridine and glu‑ cose is added to the sample and, only if blood is present, beau‑ tiful red crystals are formed. Similar crystal tests involving other reagents have also been developed (e.g., Wagenhaar and Teichmann tests). FIGURE 6.13 Forensic officer performing a blood verification Many of these tests have now been replaced with methods test on a stain on the sole of a shoe. During the test, a swab that not only determine if blood is present but also whether is treated with chemicals and then wiped over the stain. If the the blood is human. These methods usually involve a process stain is blood, the chemicals react and the swab changes color. called immunoassay. The test being used is the Kastle‑Meyer presumptive blood test. If blood is found on the shoes, its DNA can be extracted and analyzed. 6.2.4.3 Question Two: Is It Human Blood? Source: Jim Varney/Science Photo Library. Used with The method for figuring out whether a blood sample is human permission. blood or whether it comes from another animal is usually answered in a manner quite similar to the way our bodies recognize and attack invading substances, such as bacteria a series of chemical reactions that ultimately cause the luminol and viruses. When our bodies detect an invader, they swing to briefly glow in the dark through a process called chemilu‑ into action and ultimately develop antibodies very specific for minescence (emission of light from chemical reactions with‑ defeating the invader at hand, attacking only where needed. out the emission of heat). The blue glow, lasting for up to a These antibodies destroy the source of the infection and effec‑ minute, can be readily seen and photographed in a darkened tively remove it from the system. In other terms, our bodies room (Figure 6.14). This technique is very sensitive to even react to an outside antigen (a virus or bacterium, for instance) very small trace amounts of blood. Luminol has, however, and generate a specific antibody just for that one, and only, some significant problems with its use. It also glows in the antigen. An antigen is any substance that can stimulate the presence of copper, horseradish, bleach, fecal matter, certain production of antibodies, and an antibody is a Y‑shaped pro‑ plant enzymes, and animal blood. While not proven, luminol tein molecule that can combine with a foreign antigen to dis‑ is also a suspected carcinogen. able or destroy it. A quite similar process can also be used to Fluorescein is similarly applied to a suspected area, and determine if a blood sample is human or not. the area is illuminated by an ultraviolet light (often called an Our blood serum contains numerous proteins unique alternate light source, ALS). The iron of the heme group cata‑ to humans. If human blood serum is injected into a rab‑ lyzes the oxidation of the solution, which will then fluoresce bit, the rabbit’s system must identify and destroy the human 166 Introduction to Forensic Science FIGURE 6.14 After spraying a suspected bloodstain area with luminol, a glow indicates the possible presence of blood. Source: Shutterstock.com. BOX 6.3 IS IT BLOOD: THE SHROUD OF TURIN Since at least 1350, faithful pilgrims have made their way across Europe to view a piece of cloth claimed by many to be the final burial shroud of Jesus. Probably the most striking feature of this remarkable 14’ by 4’ cloth is a photographic‑quality image of a presumably crucified man imprinted in the fabric (Figure 6.15, right). Throughout its long history, there have been both strong supporters and critics of the shroud’s authenticity, including the Catholic Church’s official skepticism for hundreds of years. In recent years, a variety of scientific methods have been used to determine if the shroud is a medieval FIGURE 6.15 Images of the Shroud of Turin: (Left) one‑half of the Shroud and (Right) a close‑up of the facial region of the Shroud.  6 Forensic Serology 167 forgery or a 2,000‑year‑old relic, with mostly inconclusive and even contradictory results. Studies in the 1980s using radio‑ carbon dating methods dated the cloth to the mid‑1300s, but recent reports have shown that the fibers studied originated from a medieval patch and not from the true shroud, such that the real date of the cloth still remains to be established (Rogers, 2005). One part of the image contains what appear to be bloodstains where the nails were used for crucifixion. Several spectroscopic and presumptive tests have now indicated that the material is likely blood (Figure 6.15, left). Blood proteins and heme groups have been detected in the bloodstains but not in other regions of the cloth. While much more needs to be learned about the shroud, it does appear that, at least, the bloodstains are likely genuine. invaders (antigens) to survive. The rabbit’s body responds precipitate is noticed at the juncture of the two solutions. The by producing antibodies against the foreign human proteins, ring precipitin test, for example, simply places the anti‑human called anti‑human serum antibodies, since they specifically serum in a small test tube and the bloodstain extract is then bind to human serum proteins. If this anti‑human serum is carefully layered on top of the anti‑human serum (Figure 6.16). then removed from the rabbit, it will still attack any human The dissolved antigens and antibodies then diffuse toward each blood it encounters, causing a “precipitin reaction” to occur. As other and a precipitate is formed at the interface between the the name implies, when a precipitin reaction occurs between two solutions only if human blood is present. Variations on this an antigen and an antibody, a visible precipitate or solid will general method employ gels, glass plates, electrophoresis, and form. So, in a forensic setting, a suspected blood sample (anti‑ other methods, but the basic concept remains the same for all. gen serum) is brought into contact with anti‑human serum This same idea of using antibodies specific to a certain (from the rabbit), and if a precipitate forms, then the sample biological substance to uniquely identify it can be extended to is not only blood but must also be human blood. The process the analysis of both blood and non‑blood substances, includ‑ of using specific antibodies to identify biological samples is ing drugs and poisons. Two particularly powerful immuno‑ called immunoassay. assay techniques, called Enzyme Multiplied Immunoassay Anti‑sera can be developed in a very similar fashion for Technique (EMIT) and Enzyme Linked Immunosorbent Assay essentially any protein antigen from any animal species, as the (ELISA), have been developed for these types of analyses. antigen‑antibody reaction is a uniquely species‑specific inter‑ action. Thus, human blood will not react with chicken or horse 6.2.4.4 EMIT (Enzyme Multiplied anti‑serum, only with anti‑human serum. Several variants of the precipitin test have been devel‑ Immunoassay Technique) oped over the years to take advantage of the specificity of this In this analytical method, the biological compound or drug antigen‑antibody reaction. In general, they involve exposing a that we wish to analyze for is first attached to a protein and solution containing the human anti‑serum to a second solution then injected into an animal such as a rabbit or rat. As with containing the suspected blood sample. If there is a reaction, a any foreign protein, the animal makes antibodies to this Blood Human blood antigens in antigens in solution solution Zone of equivalence Precipitation (visible precipitate: antibody-antigen interaction) Anti-human blood Anti-human antibodies blood in solution antibodies in solution Negative Test Positive Test (no precipitation) (precipitation) FIGURE 6.16 Ring Precipitin Test. At the bottom of the test tube is the solution containing anti‑human blood antibodies in solution. The solution at the top is the test solution containing the suspected blood serum (antigen). A ring develops at the interface between these solutions from an antibody‑antigen reaction if blood is present, as shown in the center. The image on the right illustrates what happens during a positive test. 168 Introduction to Forensic Science protein‑linked antigen that can be isolated from the ani‑ how much of a particular drug is in a sample, information that mal’s serum. In the case of a drug/protein antigen, we have may be important to medicolegal investigations (see Chapters now prepared an anti‑drug serum that will react specifically 8 and 13). with the drug we are interested in. When used to determine A variant of this method is called radioimmunoassay if that particular drug is present in someone’s urine or blood, (RIA). Instead of using an enzyme linked to the drug in the for example, a sample of urine is first mixed with a sample final step, the drug is instead linked to a radioactive tracer. By of anti‑drug serum. Any drug present in the urine then com‑ measuring the radioactivity level, it is similarly possible to pletely reacts with the anti‑drug serum (antibodies). In the final determine the quantity of a specific drug or protein antigen in step, a carefully measured amount of the same drug bound to the sample. Other methods have been developed that use opti‑ an enzyme is added. Any unreacted anti‑drug serum that is cal, fluorescence, and magnetic labels to determine the amount still in the solution will react with this added “extra” drug/ of antigen/antibody binding in the EMIT experiment. enzyme complex. By measuring how much unreacted drug/ enzyme complex remains at the end of this reaction, it is pos‑ sible to determine how much of the anti‑serum was used up in reacting with the drug in the original sample, as shown in BOX 6.4 BRIEF ON CONFIRMATORY Figure 6.17. For example, if a solution contained 100 molecules AND IMMUNOASSAY BLOOD TESTS of anti‑methadone antibodies and we added to it a solution of 1. Confirmatory blood tests indicate the pres‑ urine or blood that contained 60 molecules of methadone, ence of blood in the sample with a very high all the methadone would react with the antibodies to form degree of certainty. 60 methadone/anti‑methadone complexes. This leaves, how‑ 2. Older tests involve the crystallization of blood. ever, 40 free antibodies after all the methadone is used up. If 3. Immunoassay methods capitalize on the spe‑ we now add to this another solution containing 100 molecules cific nature of antibody‑antigen reactions. of a new methadone/enzyme complex (and we know there are 4. Using specific antibodies, we can test for exactly 100 present), 40 of these enzyme complexes will react blood components or blood‑based chemicals with the remaining unused anti‑methadone antibodies left over (e.g., drugs, poisons, toxins, etc.). from the earlier reaction. This leaves 60 methadone/enzyme complexes unreacted in the solution that we can measure sim‑ ply by determining the remaining enzyme activity (only 60% of its starting activity remains if 40 have been bound to the 6.2.4.5 ELISA (Enzyme Linked drug). Thus, by some simple math, we can work backward to determine that there must have been 60 methadone molecules Immunosorbent Assay) in our original sample. So, in a conceptually similar example, Another important immunoassay technique is known as the if you gave a cashier a dollar (100 cents) and got back 60 cents ELISA method (Figure 6.18). In this method, the antibodies in change, you would know that the item that you purchased specific to a particular protein or drug are made as before but cost 40 cents without ever looking at the price tag. The entire then are attached firmly to the plastic surface of a small r­ eactor. EMIT process may be done in a single step, allowing the drug The solution containing the drug or protein (antigen) to be in the sample to compete with the drug/enzyme complex for tested for, such as methadone or blood albumin, is then added a limited amount of the specific antibodies. In this way, we to the reactor. If any of the specific drug or protein is pres‑ have an accurate and very sensitive method for determining ent in the sample, it binds tightly to the antibodies attached to the wall of the reactor. After washing away everything but the Anbody Enzyme-labeled Test Angen (limited amount) Angen tightly bound antibody and antigen, another solution containing Substrate the same antigen‑specific antibody is added, except that this E Product second antibody is attached to an enzyme – something like a + + biochemical flag. These new antibody‑enzyme complexes bind only to where a drug is already attached to an antibody stuck to the wall of the reactor. After again washing away anything that is not bound to the reactor’s walls, any antibody‑enzyme complex found remaining in the reactor must be attached to a Substrate drug‑antibody stuck to the reactor wall. We then simply look E No Product for the “flags” (or enzymes) attached to the “stuck” drug to determine how much drug (or protein) was in the original solu‑ Formed tion. Here’s a simple analogy that might be helpful. Say that we had an unknown number of darts, dipped in sugar, that we Enzyme Inacvated threw at a target in the dark (presume all hit the target). Since Complex we can’t visually see how many darts we threw, we need an indirect method to figure this out. So, we next release a group FIGURE 6.17 EMIT: Enzyme Multiplied Immunoassay Technique. of fireflies that are attracted to the sugar on the darts (say only  6 Forensic Serology 169 FIGURE 6.18 (Left) An indirect immunoassay is used to measure the concentration of antigen. The basic components of the test are antigen, labeled antibody, and substrate. (Right) A sandwich immunoassay similarly measures the concentration of antigen. The basic components of the test are antigen, labeled antibody, and substrate. Source: Images courtesy Shutterstock.com. one firefly per dart). Now, since we can “see” the glow from monoclonal antibodies has been developed to deal with this each firefly in the dark, we simply count the number of fireflies very effectively. A monoclonal antibody is an antibody that glowing, and we can determine the number of darts that were is more uniform than our natural antibodies and attacks and thrown. This is essentially what happens in ELISA – the drug/ binds to only one site on a chosen antigen. These monoclonal antigen (the dart) is fixed onto the plastic immobilized antibody antibodies can be used to protect against diseases, diagnose (the dartboard), and visualizing the second “flagged” antibody illnesses, and detect the presence of drugs and other abnormal (fireflies) allows us to determine how much drug/antigen was in chemical compounds in the blood. the sample (the number of darts fixed to the dartboard). Several Most of these methods first require the preparation of very variants of the ELISA method have also been developed. specific antibodies. Monoclonal antibody technologies allow One problem with all these analyses for drug detection is us to efficiently prepare pure antibodies in very large quanti‑ that there can be positive reactions for other compounds hav‑ ties. In summary, as shown in Figure 6.19, the antigen of inter‑ ing chemical structures similar to the drug being investigated. est (e.g., blood protein or protein‑linked drug) is injected into Both EMIT and ELISA have significant similarities and a mouse to form specific antibodies against the injected anti‑ differences. The chemicals for both the EMIT and ELISA gen. Instead of isolating the antibodies produced, as was done experiments last a long time, and the procedure can be per‑ in the previous techniques, this time we actually remove the formed with minimal training and experience. EMIT is spleen cells from the mouse that produce the antibodies them‑ primarily used for small molecules such as drugs, drug metab‑ selves. This process is like removing the factory that produces olites, and hormone analyses. ELISA measurements, however, the antibodies in the mouse’s body. These spleen cells are then are primarily used in analyzing larger molecules, such as fused with fast‑growing cancer cells to form a new hybrid cell protein antigens and antibodies, and are used for diagnosing (hybridoma). These new cells can be screened to select only infectious diseases and blood immunoglobulins. The EMIT the best producers of the antibody we want. These champion method tends to be faster, but ELISA‑based methods have producers, called “immortal cells,” are then cultured and greater sensitivity. grown to make a continuous and permanent supply chain of pure antibodies for use in immunoassays. This is analogous to creating a permanent antibody‑growing factory to supply a 6.2.4.6 Monoclonal Antibodies continuous stream of the desired antibody forever. When our bodies produce antibodies to attack an invading substance, they actually prepare a whole range of antibodies 6.2.4.7 Question Three: Whose Blood Is It? that target different parts of the invader (called polyclonal anti‑ bodies). This is similar to what real armies do; they have infan‑ The question of individualizing a biological sample uniquely try, cavalry, artillery, and other specialties that target different to one particular person has long been a central goal. Blood parts of the same enemy. While this works well for armies and analysis held out the first real hope that this type of specific our bodies, it can also cause significant problems if we want connection was possible; a hope that was ultimately fulfilled antibodies that can reliably target just one particular molecule by DNA typing. Nonetheless, individual information, at times for the immunoassay techniques already described. Luckily, approaching the specificity of DNA typing, has been achieved a powerful technique involving the use of something called through careful blood typing studies. 170 Introduction to Forensic Science FIGURE 6.19 Hybridoma technology of monoclonal antibody production. Source: Shutterstock.com. In 1901, Karl Landsteiner discovered what we know sugar components attached) that is found sticking to the sur‑ today as blood groups. His immediate concern at the time face of RBCs. These glycoproteins contain one of three differ‑ was to find a safe and reliable way to know when blood from ent chemical ends, just like “snap‑on” attachments that fit onto a donor can be safely given intravenously to a patient in need. the end of a tool. The “basic” version of the glycoprotein ends He found that blood of one type of donor, which he called type with a certain sugar (fructose) exposed, and this entire unit A, could be safely mixed with some blood samples without is referred to as the H antigen (Figure 6.20). The presence of agglutination (clumping) but not with others, for example type only the H antigen on the RBC gives rise to the O blood type. B donors. Type B donors, in turn, could be mixed with blood If we “snap‑on” an additional sugar (acetylgalactose amine) to from other type B donors without problems but could not be the end of the H antigen, the A antigen is formed, which gives mixed with type A. Further experiments allowed him to ulti‑ rise to the A blood type. If instead, we “snap‑on” a different mately classify blood into the four different types we know sugar (galactose), then the B antigen is formed, which leads to today as the ABO system. the B blood type. Since we inherit one gene from our mothers and one from our fathers, six possible combinations from these three antigens are possible: HH, HA, AA, HB, BB, and AB. BOX 6.5 BRIEF ON BLOOD TYPING Since A and B are genetically co‑dominant (have the same genetic “strengths” for inheritance), these six possibilities lead 1. Different proteins (antigens) are present on to the four observed blood types in the ABO system: A (AA or the surface of RBCs and are inherited. AH), B (BB or BH), AB (AB only), and O (HH). This is shown 2. The presence or absence of different red blood schematically in Figure 6.20 and summarized in Table 6.1. cell (RBC) antigens leads to different blood As we’ve learned already, blood plasma contains sub‑ types. stances called antibodies that target very specific proteins, 3. The most commonly employed blood groups such as the glycoproteins found on the surface of our RBCs. involve the ABO and Rh systems. Within the plasma of a person with type A blood is contained 4. Agglutination occurs when antibodies in the antibodies against the B antigen (anti‑B). These antibodies are plasma specific for an RBC antigen react to present even before there is contact with B type blood, possibly “clump” cells together. due to the fact that some bacteria and plants also have these 5. Four possible blood types exist in the ABO antigens so that we develop antibodies to them from a very blood system (A, B, AB, and O). young age. Similarly, the plasma of a person who carries the 6. More than 600 RBC antigens are known. B antigen on their RBCs (B type blood) contains antibodies against the A antigen (anti‑A). If we mix type A blood sam‑ ples with plasma from a person with B type blood, an immune Our DNA carries a gene that dictates the formation of reaction will occur and the RBCs will clump together in a pro‑ one of three possible forms of a glycoprotein (a protein with cess called agglutination. This is shown in Figure 6.21. Note  6 Forensic Serology 171 FIGURE 6.20 The antigens giving rise to the different ABO blood types. In O type blood, there is no “additional” sugar cap on the end of the glycoprotein. In types A and B, there are other sugars capping the H‑end sugar. In type AB, both A and B sugars are pres- ent. Note that there are two genotypic ways to have either type A and type B blood. TABLE 6.1 Blood type antigens and antibodies END GROUP (ATTACHMENT) ANTIBODY PRESENT IN BLOOD FREQUENCY (%) GENOTYPE N‑acetylgalactosamine Anti‑B 42 AA or AO D‑galactose Anti‑A 10 BB or BO Both N‑acetylgalactosamine and d‑galactose None 4 AB L‑fructose Both anti‑A and anti‑B 44 OO Antibodies Agglutination acceptor blood type since it does not contain antibodies against either the A antigen or the B antigen. These people can usu‑ ally receive RBCs from any of the ABO blood types without Red problems. Type O blood is called the universal donor since it Blood Cell contains neither A nor B antigens on the RBCs, while type AB is the universal donor for blood plasma. There is no known antibody against the H antigen. Antigens Determining the ABO blood type of a particular sample simply requires mixing the sample blood separately with FIGURE 6.21 The drawing on the left shows two red blood anti‑A and anti‑B sera. If the A antigen is present in the blood cells with the surface antigens (small green shapes) and the yel- sample (from either type A or AB blood), then an agglutination low serum antibodies (small diamonds) indicated. The antibod- reaction occurs when mixed with Anti‑A serum. If the B anti‑ ies bind with the antigen, causing the red blood cells to clump gen is present (from either type B or AB blood), then no reac‑ together through agglutination. tion occurs with the anti‑A serum but agglutination does occur when mixed with anti‑B serum. If no reaction is observed with that agglutination refers to the clumping of RBCs together due either anti‑A or anti‑B sera, then the blood is type O. Examples to antigen‑antibody reactions while coagulation, or clotting, of this process are shown in Figure 6.22. is the process of converting blood into a jelly‑like substance The ABO system is only one of at least 29 known blood from the action of activated thrombocytes sticking blood cel‑ group systems. A complete blood type description for a blood lular material together. A sample of type O plasma contains sample, although very rarely done, would potentially include antibodies against both A and B (anti‑A and anti‑B), while an analysis for the full set of substances found on the surface type AB blood plasma contains neither of these antibodies. of the RBCs. In the 29 blood group systems, well over 600 This is why type AB blood is often referred to as the universal different blood antigens are known. A person’s complete blood 172 Introduction to Forensic Science FIGURE 6.22 The blood group test card for a type A+ blood sample. The blood grouping card shows agglutination of blood with anti‑A and anti‑Rh(D), but not with anti‑B. Source: Used per Creative Commons Attribution 2.0 Generic, User: Apers0n at English Wikimedia. Note: D refers to the Rh factor. FIGURE 6.23 Distribution of the type O blood in native populations of the world. Source: Used per Creative Commons Attribution Share‑Alike 3.0, Unported, User: anthro palomar. type would be one of the many, many possible combinations populations for two blood group systems (ABO and Rhesus) of these blood group antigens. Many of these combinations, is given in Table 6.2, and the variation in the frequency of the however, are very rare or limited to certain ethnic groups. The O blood type around the world is shown in Figure 6.23. The ethnic distribution of particular blood antigens can, in some rarest blood type known is the “Bombay” type that is present instances, provide useful forensic information. For example, in about 0.0004% of the population (about four people in a one Rhesus antigen is present in less than 0.5% of Caucasians million) (the Bombay type is missing the H antigen entirely). but present in 40% of West Africans. In contrast, the Kell anti‑ Besides the ABO system, the other most common blood gen is essentially only found in Caucasians, and the Diego anti‑ group is the Rhesus blood group (Rh). Named for its first isola‑ gen is absent from Caucasians yet present in blood from most tion from the Rhesus monkey, blood labeled Rh positive con‑ Japanese and Chinese people. The percentage of two different tains the D antigen on the surface of RBCs, while Rh‑negative  6 Forensic Serology 173 TABLE 6.2 Blood type frequencies BLOOD TYPE (ABO AND RH) POPULATION % IN S. KOREA POPULATION % IN US A positive 34.4 35.7 A negative 0.1 6.3 B positive 26.8 8.5 B negative 0.1 1.5 AB positive 11.2 3.4 AB negative 0.05 0.6 O positive 27.4 37.4 O negative 0.1 6.6 blood lacks the D antigen. In typical blood transfusion work, TABLE 6.3 ABO blood type allele contributions the ABO and Rh systems are both determined to establish the FATHER’S ALLELE compatibility of the blood. CONTRIBUTION A B O 6.3.5 Blood Type Inheritance Mother’s allele contribution A AA AB AO (type A) (type AB) (type A) and Parental Testing B BA BB BO (type AB) (type B) (type B) Since blood types are determined through the actions of our O OA OB OO genes, blood types are inherited from our parents and do not (type A) (type B) (type O) change throughout our lives. A person’s ABO blood type, for example, is determined by which alleles they inherit: the H (often just called O), A, or B antigens. Both the A and B alleles Blood typing can help identify the father in paternity cases, are dominant over O and co‑dominant to each other (co‑dom‑ usually by conclusively ruling out possible fathers (Table 6.3). inant means equal genetic “strength” in determining inheri‑ For example, two type O parents cannot have a type A or B tance). This means that when only one O allele is present, the child. Similarly, two type A parents cannot have a type B or AB other allele dictates the blood type. A person who is AO will child. Adding other blood tests, such as Rh, MN, and others, have an A blood type. It takes the combination of two O alleles can provide a greater certainty in the result. The final positive to produce the O blood type. The combination of one A and determination of paternity in the United States and most other one B allele results in the AB blood type. This is summarized countries is no longer, however, established on blood typing in Table 6.3. but is now based on DNA typing. According to the American The Rh system is far more complex than the ABO system, Association of Blood Banks, 27.9% of men accused of pater‑ with over 35 different possibilities from each parent. Generally, nity that are tested are not the biological fathers (see Box 6.6). these possibilities fall into what are referred to as positive or In addition, about 15% of men named on a birth certificate are negative groups (see above), with the positive dominant over not the biological fathers based on DNA typing. Many states the negative. Because of the complexity of this grouping, how‑ assume that a woman’s husband is the father, allowing her to sue ever, it is possible for two Rh‑positive parents to produce an for child support. The accused father then carries the burden of Rh‑negative child. proof to show that he is not the true father.